title: Catalytic and FAD-binding residues of mitochondrial very long chain acyl-coenzyme A dehydrogenase.
abstract: Very long-chain acyl-CoA dehydrogenase (VLCAD) is one of four flavoproteins which catalyze the initial step of the mitochondrial beta-oxidation spiral. By sequence comparison with other acyl-CoA dehydrogenases, Glu-422 of VLCAD has been presumed to be the catalytic residue that abstracts the alpha-proton in the alphabeta-dehydrogenation reaction. Replacing Glu-422 with glutamine (E422Q) caused a loss of enzyme activity by preventing the formation of a charge transfer complex between VLCAD and palmitoyl-CoA. This result provides further evidence for Glu-422 being part of the active site of VLCAD. F418L is a disease-causing mutation in human VLCAD deficiency. Unlike wild-type VLCAD, F418L and F418V contained no bound FAD when expressed at extremely high levels in the baculovirus expression system. Although F418T and F418Y bound FAD at a level similar to that of wild-type VLCAD, both showed reduced Vmax values toward palmitoyl-CoA, most likely due to a decrease in the rate of enzyme-bound FAD reduction. These data suggest that Phe-418 is involved in the binding and subsequent reduction of FAD. FAD-deficient VLCADs (F418L, F418V, and apo-VLCAD) showed increased sensitivity to trypsinization. Loss of FAD may change the folding of VLCAD subunit.
authors.affiliation: Department of Biochemistry, Shinshu University School of Medicine, Matsumoto, Nagano 390, Japan. msouri@med.id.yamagata-u.ac.jp
authors.forename: M
authors.initials: M
authors.lastname: Souri
authors.forename: T
authors.initials: T
authors.lastname: Aoyama
authors.forename: G F
authors.initials: GF
authors.lastname: Cox
authors.forename: T
authors.initials: T
authors.lastname: Hashimoto
mesh_terms: D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D010171:Palmitoyl Coenzyme A; D044942:Acyl-CoA Dehydrogenase, Long-Chain; D014357:Trypsin